Positive controls

ABSTRACT

The present invention relates to the use of one or more amplicons as temperature calibrators. In some embodiments, the calibrators may be used to calibrate the temperature of a microfluidic channel in which amplification and/or melt analysis is performed. In some embodiments, the amplicons may be genomic, ultra conserved elements and/or synthetic. The amplicon(s) may have a known or expected melt temperature(s). The calibrators may be added to primers of study or may follow or lead the primers of study in the channel. The amplicon(s) may be amplified and melted, and the temperature(s) at which the amplicon(s) melted may be determined. The measured temperature(s) may be compared to the known temperature(s) at which the amplicon(s) was expected to melt. The difference(s) between the measured and expected temperatures may be used to calibrate/adjust one or more temperature control elements used to control and/or detect the temperature of the channel. In other embodiments, the UCE primers may function as a positive control to validate amplification has occurred.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of the U.S.application Ser. No. 13/223,258 claiming the benefit of priority to U.S.Provisional Application Ser. No. 61/378,927, filed on Aug. 31, 2010, theentire disclosure of which is incorporated herein by reference. Inaddition, the present application claims the benefit of priority to U.S.Provisional Application Ser. No. 61/378,591, filed on Aug. 31, 2010, theentire disclosure of which is incorporated herein by reference.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled3400233SequenceListing.txt, was created on Aug. 30, 2011 and is 11 kb insize. The information in the electronic format of the Sequence Listingis part of the present application and is incorporated herein byreference in its entirety.

BACKGROUND

Field of the Invention

The present invention relates to methods, devices, and systems forcalibrating a temperature of one or more microfluidic channels in amicrofluidic device. More particularly, aspects of the present inventionrelate to methods, devices, and systems for using an amplicon having aknown or expected thermal melting temperature to calibrate a thermalsensor and/or thermal control element used to control the temperature ofa microfluidic channel in a microfluidic device. The present inventionalso relates to methods, devices and systems for calibrating thetemperature of a thermal melt in a microfluidic channel of amicrofluidic device. More particularly, aspects of the present inventionrelate to methods, devices, and systems for using a calibrant present ina test sample and amplifying the calibrant to produce a calibrantamplicon in which the calibrant amplicon has a known or expected thermalmelting temperature to calibrate the melting temperature of an ampliconof a nucleic acid of interest. The present invention also relates to theuse of a calibrant present in a test sample as a control for determiningwhether amplification of a sample has occurred.

Description of the Background

Devices for performing chemical, biological, or other reactions (e.g., amicrofluidic device for performing polymerase chain reaction (PCR)amplification of DNA molecules, or a microfluidic molecular diagnosticplatform that performs PCR on a patient sample and then uses the PCRproduct for genotyping by performing a high resolution melt analysis)often feature one or more thermal control elements that are used tosubject reactants to a desired thermal profile. A description of PCRamplification, and an example of one possible microfluidic deviceincluding thermal control elements for PCR amplification and thermalmelt analysis, are provided in U.S. Patent Application Publication Nos.2009/0248349 and 2011/0056926, the entire disclosures of which areincorporated herein by reference.

In many applications of such microfluidic devices (e.g., PCR and/orthermal melt analysis), the thermal control elements of those devicesmust be precisely calibrated. That is, the correspondence between thetemperature of the thermal control element and an electricalcharacteristic of the thermal control element must be preciselydetermined. For example, in the case of a resistance temperaturedetector and/or heater, the correspondence between temperature andresistance must be precisely determined. Additional types of thermalcontrol elements can include platinum resistive heaters, thermistors,diode temperature sensors, thermocouples, or any other suitabletemperature measuring devices. Additional electrical characteristics ofthermal control elements that correspond to temperature can includecapacitance or inductance of an element, frequency, pulse width, oramplitude of a signal, or other sensor characteristics known in the art.

Among many variations that can occur in microfluidic devices,temperature variation between channels is particularly relevant.Securing the right and uniform temperature among channels in amicrofluidic device or chip leads to a reliable and consistent PCR andthermal melt analysis. In turn, having a successful amplification andmelt of the target sequence may lead to a correct sample genotyping,which may lead to the characterization of a patient with a geneticdisorder or a genetic predisposition or the efficiency of drugmetabolism.

As noted above, assuring that microfluidic channels in a chip presentthe same temperature during a PCR and thermal melt analysis reactionwill result in improved reliability and consistency. However, with suchsmall elements and such small volumes of reagents, it is verychallenging to test and prove that channels at the same time areperforming a PCR plus thermal melting under the same optimal temperatureconditions.

Methods of calibrating thermal control elements of a microfluidic deviceoften include generating a lookup table or a series of coefficients thatdefine a calibration equation, i.e., a lookup table or an equationrelating the temperature of the thermal control element with theelectrical characteristic.

Calibration can be performed by sending the device to a third partylaboratory for taking accurate measurements and generating the lookuptable or series of coefficients; however, this procedure is generallyexpensive and time consuming. Furthermore, for many devices (e.g., manycommon microfluidic devices) there may be many thermal control elements(e.g. dozens or even hundreds of heaters and sensors), each of whichrequires its own precise calibration, making third-party calibrationimpractical.

Accordingly, there is a need in the art for a reliable calibrant thatwill be useful in calibrating the temperature of one or moremicrofluidic channels in a microfluidic device or chip. Similarly, thereis a need for robust calibration of thermal sensors and/or heaters thatcan be accurate, reduce downtime and maintain high throughput.

SUMMARY

In one aspect, the present invention provides a method of calibratingthe temperature of a microfluidic channel present in a microfluidicsystem comprising a controller. The method may comprise: (a) introducinginto the microfluidic channel reagents including one or more amplicons;(b) melting the one or more amplicons and determining a meltingtemperature of each of the one or more amplicons; (c) comparing thedetermined melting temperature of each of the one or more amplicons witha respective expected melting temperature of each of the one or moreamplicons; and (d) adjusting the controller based on the comparison ofthe determined melting temperature of each of the one or more ampliconswith the respective expected melting temperature of each of the one ormore amplicons.

In some embodiments, one or more of the one or more amplicons areprepared synthetically and then introduced into the microfluidicchannel. In some embodiments, the one or more amplicons may be preparedfrom genomic DNA. In some embodiments, the amplicons may be preparedfrom Ultra Conserved Elements (UCEs). In other embodiments, theamplicons may be prepared from any coding sequences, such as sickle cellcoding sequence. In other embodiments, the one or more amplicons may beprepared from any synthetic DNA. In additional embodiments, the one ormore amplicons may be prepared from both genomic DNA or synthetic DNA.In some embodiments, one of the one or more amplicons may be preparedfrom RFCAL-100. In other embodiments, one of the one or more ampliconsmay be prepared from RFCAL-200. In additional embodiments, the ampliconsmay be prepared from both RFCAL-100 and RFCAL-200. The controller maycontrol and/or detect the temperature of the microfluidic channel.

In one embodiment, the microfluidic channel reagents may include atleast two amplicons; the melting and determining may comprise meltingthe two or more amplicons and determining a melting temperature of eachof the two more amplicons; the comparing may comprise comparing thedetermined melting temperature of each of the two or more amplicons witha respective expected melting temperature of each of the two or moreamplicons; and the adjusting may comprise adjusting the controller basedon the comparison of the determined melting temperature of each of thetwo or more amplicons with the respective expected melting temperatureof each of the two or more amplicons. The two or more amplicons may haveknown melting temperatures more than 5 degrees Celsius apart. The two ormore amplicons may have known melting temperatures more than 10 degreesCelsius apart. The two or more amplicons may have known meltingtemperatures more than 15 degrees Celsius apart. The two or moreamplicons have known melting temperatures more than 20 degrees Celsiusapart.

In some embodiments, the determining the melting temperature of each ofthe two or more amplicons may comprise: obtaining a derivative of a meltcurve of the melting of the two or more amplicons using a Savitzky-Golayfilter, and finding the temperatures at the maximum negative derivativesof the derivative of the melt curve. The determining the meltingtemperature of each of the two more amplicons may comprise:cross-correlating peaks of a derivative of a reference melt curveagainst peaks of a derivative of a melt curve of the melting of the twoor more amplicons, and selecting the temperatures having the highestcorrelation as the melting temperatures of the two more amplicons. Thecomparing the determined melting temperature of each of the two or moreamplicons with the respective expected melting temperature of each ofthe two or more amplicons may comprise calculating a slope correctionfactor and an intercept correction factor using the respective expectedmelting temperature of each of the two or more amplicons. The adjustingthe controller may comprise calculating an adjusted temperature usingthe calculated slope correction factor and the calculated interceptcorrection factor.

In one embodiment, a derivative of a melt curve of the melting of theone or more amplicons may exhibit a single peak, for instance where onlyone amplicon is used. The melting of the one or more amplicons maycomprise a first melting of the one or more amplicons and a secondmelting of the one or more amplicons. The determining the meltingtemperature of each of the one or more amplicons may comprise (i)determining a first temperature corresponding to a peak of thederivative of the melt curve of the first melting of the one or moreamplicons and (ii) determining a second temperature corresponding to apeak of the derivative of the melt curve of the second melting of theone or more amplicons. The adjusting the controller may comprisecalculating a temperature adjustment using a linear interpolation of thefirst and second temperatures.

In another aspect, the present invention provides a method ofcalibrating the temperature of a thermal melt in a microfluidic channelpresent in a microfluidic system. The method may comprise: (a)introducing into the microfluidic channel reagents comprising a testsample containing human genomic DNA, at least a pair of primers for oneor more nucleic acids of interest and a pair of primers for a calibrant,wherein the calibrant is found in genomic DNA; (b) amplifying thegenomic DNA to produce a calibrant amplicon and an amplicon of the atleast one nucleic acid of interest; (c) melting the amplicons anddetermining a melting temperature of each of the amplicons; (d)comparing the determined melting temperature of the calibrant ampliconwith an expected melting temperature of the calibrant amplicon; and (e)calibrating the melting temperature of the amplicon of the at least onenucleic acid of interest based on the melting temperature of thecalibrant amplicon.

It is within the scope of the invention that segments of fluid may beadded to the microfluidic channel sequentially. Each segment of fluidmay be of different composition than a segments directly adjacent to it.In one embodiment, the pair of primers for the calibrant may be added tothe microfluidic channel in a separate fluid segment (“calibrant fluidsegment”) than the at least one pair of primers for the one or morenucleic acids of interest (“assay fluid segment”). In some embodiments,one or more calibrant fluid segments may be added to the microfluidicchannel prior to multiple assay fluid segments being added to themicrofluidic channel. In other embodiments, one or more calibrant fluidsegments may be added to the microfluidic channel before multiple assayfluid segments are added to the microfluidic channel, and one or morecalibrant fluid segments may be added after the assay fluid segments. Inother embodiments, calibrant fluid segments may alternate with assayfluid segments such that each assay fluid segment has a calibrant fluidsegment adjacent on either side. It is therefore noted that throughoutthe specification, the introduction of at least one pair of primers forthe one or more nucleic acids of interest and the introduction of thepair of primers for the calibrant into the microfluidic channel canoccur in the same or in separate fluid segments, wherein the separatefluid segments may be introduced in any order, alternately,sequentially, or at the start and end of multiple fluid segments.

In some embodiments, the microfluidic channel reagents include a pair ofprimers for one nucleic acid of interest. As used herein, nucleic acidof interest refers to a nucleic acid whose presence or absence is to bedetermined in a test sample. In other embodiments, the microfluidicchannel reagents include a pair of primers for each of two or morenucleic acids of interest. In some embodiments, the calibrant found ingenomic DNA is a DNA segment that is known to be present in all genomicDNA of interest, such as human genomic DNA. In other embodiments, thecalibrant is a UCE. In some embodiments, the microfluidic channelreagents may contain a pair of primers for the calibrant. In certainembodiments, the amplification of the microfluidic reagents produce acalibrant amplicon. In certain embodiments, the amplification of thecalibrant dos not affect amplification of the one or more nuclei acidsof interest.

In some embodiments, the determining the melting temperature of each ofthe amplicons may comprise: obtaining a derivative of a melt curve ofthe melting of the amplicons using a Savitzky-Golay filter, and findingthe temperatures at the maximum negative derivatives of the derivativeof the melt curve. The determining the melting temperature of each ofthe amplicons may comprise: cross-correlating peaks of a derivative of areference melt curve against peaks of a derivative of a melt curve ofthe melting of the amplicons, and selecting the temperatures having thehighest correlation as the melting temperatures of the amplicons. Thecomparing the determined melting temperature of the calibrant ampliconwith the respective expected melting temperature of the calibrantamplicon may comprise calculating a slope correction factor and anintercept correction factor using the respective expected meltingtemperature of the calibrant amplicon. Calibrating the meltingtemperature of each of the amplicons of the nucleic acids of interestmay comprise calculating an adjusted temperature using the calculatedslope correction factor and the calculated intercept correction factorof the calibrant amplicon.

The method may comprise using the comparison of the determined meltingtemperature of the calibrant amplicon with the respective expectedmelting temperature of the calibrant amplicon to validate amplificationof the one or more amplicons of the one or more nucleic acids ofinterest. The validation may comprise determining the corrected meltingtemperatures of the amplicons of the nucleic acids of interest.

The method may also comprise using the melt curve of the calibrantamplicon as a control to validate amplification has occurred. Obtaininga melt curve for the calibrant can serve as a positive control todemonstrate that no contamination of the reagents or other anomalieshave occurred that would prevent amplification of the genomic DNA.

In another aspect, the present invention provides a method ofcalibrating the temperature of a microfluidic channel. The method maycomprise: performing in the microfluidic channel a thermal melt analysisof at least one amplicon having a known thermal melting temperature andadjusting a thermal control element based on a deviation of the thermalmelting temperature from the known value.

In still another aspect, the present invention may provide a calibrantcomprising RFCAL-100.

In yet another aspect, the present invention may provide a calibrantcomprising RFCAL-200. The calibrant may further comprise RFCAL-100.

In another aspect, the present invention provides a calibrant (a) whichis present in human genomic DNA, (b) amplification of which produces anamplicon having a known melting temperature that does not interfere withthe amplification of one or more nucleic acids of interest that may bepresent in the human genomic DNA.

In one aspect of the invention, a system and a method for a positivecontrol of a biological reaction are provided. Specifically, thepositive control may be based on the usage of ultra-conserved elements(UCE) in the biological reaction. A device is configured to receive abiological sample to perform a biological reaction. A control sample anda test sample, each of which comprises reagents for the biologicalreaction, are provided. Both the control and the test sample comprisebiological material from the sample to be tested. The control samplewill contain reagents specific to the UCE, such as primers. The controland the test samples are loaded into the desired device and thebiological reaction is run on the test sample and on the control sample.Furthermore, a controller is configured to analyze a reaction result forthe test sample using the control sample as a positive control.Specifically, if the control sample produces an expected result thebiological reaction on the test sample is determined to have beensuccessful. If the control sample produces an unexpected result, thebiological reaction on the test sample is determined to have beenunsuccessful.

In one embodiment, the biological reaction is performed separately oneach of the test and control samples. In yet another embodiment, thecontrol sample is combined with the test sample and a single biologicalreaction is run on the sample including both the test and controlmaterial. In one embodiment, the biological reaction may be anyamplification, thermal melt, or other nucleic acid processing reaction.In the case of a thermal melt or nucleic acid processing reaction, thecontrol sample comprising UCE is used to validate the test sample, whichmay include the products of a previous amplification of the test nucleicacids. In the case of an amplification reaction, the test sample maycomprise test sample and test primers while the control sample maycomprise test sample (necessarily containing the UCE) and UCE primers.An expected result from the control sample resulting in UCEamplification indicates a successful sample preparation and subsequentamplification of the test sample.

In another embodiment, reaction components specific to UCEs may be usedas a positive control in combination with any biological reaction on anybiological sample. An expected result from the positive control (i.e.,the UCE) will confirm that the reaction components and equipment arefunctioning properly, regardless of the outcome of the test sample. Forinstance, such an embodiment would be useful when testing for thepresence of a particular organism in a sample. Running a control usingUCE specific reagents will allow for a determination of whether the testwas successful, regardless of whether the organism of interest waspresent in the test sample.

The above and other aspects and features of the present invention, aswell as the structure and application of various embodiments of thepresent invention, are described below with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) ofthe reference number identifies the drawing in which the referencenumber first appears.

FIG. 1 illustrates a microfluidic device embodying aspects of thepresent invention.

FIG. 2 is a functional block diagram of a system for using amicrofluidic device embodying aspects of the present invention.

FIG. 3A is a flow chart illustrating a process for calibrating thetemperature of a microfluidic channel of a microfluidic device inaccordance with an embodiment of the present invention.

FIG. 3B is a flow chart illustrating a process for calibrating thetemperature of a thermal melt in a microfluidic channel present in amicrofluidic system in accordance with an embodiment of the presentinvention.

FIG. 4 illustrates thermal melt curves of an exemplary Ultra ConservedElement (UCE) calibrant in accordance with an embodiment of theinvention on a microfluidic instrument showing three different genomicDNA.

FIG. 5 illustrates thermal melt curves of an exemplary UCE calibrant inaccordance with an embodiment of the invention on a Roche LightCycler®480 (LC480) cycler platform showing seven different genomic DNA.

FIG. 6 illustrates thermal melt curves showing three different genotypesof a primer of study.

FIG. 7 illustrates thermal melt curves showing UCE products inaccordance with an embodiment of the invention.

FIG. 8 illustrates a thermal melt curve showing three differentgenotypes.

FIG. 9 illustrates a thermal melt curve showing a UCE amplificationproduct in accordance with an embodiment of the invention.

FIG. 10 illustrates a thermal melt curve showing an amplificationproduct of a UCE primer and a primer of study in accordance with anembodiment of the invention.

FIG. 11 illustrates a thermal melt curve showing the amplificationproduct of the primer of study only.

FIG. 12 illustrates a diagram depicting a pGOv4 vector map in relationto a synthetic sequence in accordance with an embodiment of theinvention.

FIG. 13 illustrates a graph depicting the calibrators' productamplification on a LC480 cycler platform in accordance with anembodiment of the invention.

FIG. 14 illustrates a graph depicting the calibrators' product meltingon a LC480 cycler platform in accordance with an embodiment of theinvention.

FIG. 15 illustrates a graph depicting the results of RFCAL-100 on amicrofluidics-based analysis platform in accordance with an embodimentof the invention.

FIG. 16 illustrates a graph depicting the results of RFCAL-200 on amicrofluidics-based analysis platform in accordance with an embodimentof the invention.

FIG. 17 illustrates a graph depicting the RFCAL-melt profile on IdahoTechnologies HR-1 high resolution melter in accordance with anembodiment of the invention.

FIG. 18A illustrates fluorescence versus temperature melt curves forWarfarin CyP2C9*2.

FIG. 18B illustrates derivative of fluorescence with respect totemperature melt curves for Warfarin CyP2C9*2.

FIG. 19A illustrates fluorescence versus temperature melt curves for acalibrant according to an embodiment of the invention on a well platebased system, and FIG. 19B illustrates that the derivative offluorescence with respect to temperature melt curves for the UCEcalibrant exhibits a single peak.

FIG. 20A illustrates fluorescence versus temperature melt curves for acalibrant according to an embodiment of the invention on a microfluidicchannel based system, and FIG. 20B illustrates that the derivative offluorescence with respect to temperature melt curves for the calibrantexhibits two peaks.

FIG. 21A illustrates calculation of the relative temperature shift of anexperimental calibrant relative to reference calibrant from a calibratedinstrument in accordance with an embodiment of the invention.

FIG. 21B illustrates calculation of the temperature shift on theexperimental calibrant as the shift required to maximize the correlationwith the reference calibrant in accordance with an embodiment of theinvention.

FIG. 22A is a plot illustrating melting temperatures of assays of thesame genotype and interlaced UCEs collected from 7 different channels.

FIG. 22B is a plot illustrating the standard deviation of the assaysmelted at the same time across the 7 channels in FIG. 22A.

FIG. 22C is a plot illustrating the changes in the melt temperatures ofthe assays relative to the interpolated UCE melt temperature using theimmediately preceding and immediately following UCE.

FIG. 22D is a plot illustrating the corresponding standard deviation ofthe changes in the melt temperatures shown in FIG. 22C.

FIG. 22E is a plot illustrating the changes in the melt temperatures ofthe assays relative to the interpolated UCE Tm using only the first andlast UCE.

FIG. 22F is a plot illustrating the corresponding standard deviation ofchanges in the melt temperatures of the assays shown in FIG. 22E.

FIG. 23A is a plot illustrating a reference dual peak thermal melt curvefrom a calibrated instrument.

FIG. 23B is a plot illustrating an out of calibration dual peakexperimental thermal melt curve.

FIG. 23C is a plot illustrating the out of calibration dual peakexperimental thermal melt curve shown in FIG. 23B lined up with thereference dual peak thermal melt curve shown in FIG. 23A.

FIG. 23D is a plot illustrating a subsequent melt curve of anindependent calibration check melt.

FIG. 23E is a schematic diagram illustrating the functioning of theautomatic calibration procedure in accordance with an embodiment of theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 illustrates a microfluidic device 100 embodying aspects of thepresent invention. As described herein, the microfluidic device 100 mayalso be referred to interchangeably as the reaction chip or as theU-chip. In the illustrated embodiment, the microfluidic device 100includes several microfluidic channels 102 extending across a substrate101. Each channel 102 includes one or more inlet ports 103 (theillustrated embodiment shows two inlet ports 103 per channel 102) andone or more outlet ports 105 (the illustrated embodiment shows oneoutlet port 105 per channel 102). In exemplary embodiments, each channelmay be subdivided into a first portion extending through a PCR thermalzone 104 (as described below) and a second portion extending through athermal melt zone 106 (as described below).

In an embodiment, the microfluidic device 100 further includes thermalcontrol elements in the form of thin film resistive heaters 112associated with the microfluidic channels 102. In one non-limitingembodiment, the thin film resistive heaters 112 may be platinumresistive heaters whose resistances are measured in order to controltheir respective temperatures. In the embodiment illustrated in FIG. 1,each heater element 112 comprises two heater sections: a PCR heater 112a section in the PCR zone 104, and a thermal melt heater section 112 bin the thermal melt zone 106.

In one embodiment, the microfluidic device 100 includes a plurality ofheater electrodes 110 connected to the various thin-film heaters 112 aand 112 b. In non-limiting embodiments, heater electrodes 110 mayinclude PCR section leads 118, one or more PCR section common lead 116a, thermal melt section leads 120, and one or more thermal melt sectioncommon lead 116 b. According to one embodiment of the present invention,a separate PCR section lead 118 is connected to each of the thin-filmPCR heaters 112 a, and a separate thermal melt section common lead 1120is connected to each of the thin-film thermal melt heaters 112 b.

FIG. 2 illustrates a functional block diagram of a system 200 for usinga microfluidic device 100, in accordance with one embodiment. The DNAsample is input in the microfluidic chip 100 from a preparation stage202. As described herein, the preparation stage 202 may also be referredto interchangeably as the pipettor system or as the pre-K stage. Thepreparation stage 202 may comprise appropriate devices for preparing thesample 204 and for adding one or more reagents 206 to the sample. Oncethe sample is input into the microfluidic chip 100, e.g., at an inputport 103, the sample flows through a channel 102 into the PCR zone 104where PCR takes place. That is, as explained in more detail below, asthe sample flows within a channel 102 through the PCR zone 104, thesample is exposed to the PCR temperature cycle a plurality of times toeffect PCR amplification. Next, the sample flows into the thermal meltzone 106 where a high resolution thermal melt process occurs. Flow ofsample into the microfluidic chip 100 can be controlled by a flowcontroller 208. The flow controller may be part of a control system 250of the system 200. The control system 250 may comprise the flowcontroller 208, a PCR zone temperature controller 210, a PCR flowmonitor 218, a thermal melt zone temperature controller 224, and/or azone fluorescence measurement system 232.

The temperature in the PCR zone 104 can be controlled by the PCR zonetemperature controller 210. The PCR zone temperature controller 210,which may be a programmed computer or other microprocessor, sendssignals to the heater device 212 (e.g., a PCR heater 112 a) based on thetemperature determined by a temperature sensor 214 (such as, forexample, an RTD or thin-film thermistor, or a thin-film thermocouplethermometer). In this way, the temperature of the PCR zone 104 can bemaintained at the desired level. According to some embodiments of thepresent invention, the PCR zone 104 may also be cooled by a coolingdevice 216 (for example, to quickly bring the channel temperature from95° C. down to 55° C.), which may also be controlled by the PCR zonetemperature controller 210. In one embodiment, the cooling device 216could be a peltier device, heat sink or forced convection air cooleddevice, for example.

The flow of sample through the microfluidic channels 102 can be measuredby a PCR zone flow monitoring system 218. In one embodiment, the flowmonitoring system can be a fluorescent dye diffusion imaging andtracking system illustrated in U.S. Pat. No. 7,629,124, which isincorporated herein by reference in its entirety. According to oneembodiment of the present invention, the channels in the PCR zone can beexcited by an excitation device 220 and light fluoresced from the samplecan be detected by a detection device 222. An example of one possibleexcitation device and detection device forming part of an imaging systemis illustrated in U.S. Patent Application Publication No. 2008/0003593and U.S. Pat. No. 7,629,124, which are incorporated herein by referencein their entirety.

The thermal melt zone temperature controller 224, e.g. a programmedcomputer or other microprocessor, can be used to control the temperatureof the thermal melt zone 106. As with the PCR zone temperaturecontroller 210, the thermal melt zone temperature controller 224 sendssignals to the heating component 226 (e.g., a thermal melt heater 112 b)based on the temperature measured by a temperature sensor 228 which canbe, for example, an RTD or thin-film thermocouple. Additionally, thethermal melt zone 106 may be independently cooled by cooling device 230.The fluorescent signature of the sample can be measured by the thermalmelt zone fluorescence measurement system 232. The fluorescencemeasurement system 232 excites the sample with an excitation device 234,and the fluorescence of the sample can be detected by a detection device236. An example of one possible fluorescence measurement system isillustrated in U.S. Patent Application Publication No. 2008/0003593 andU.S. Pat. No. 7,629,124, which are incorporated herein by reference intheir entirety.

In accordance with aspects of the present invention, the thin filmheaters 112 may function as both heaters and temperature detectors.Thus, in one embodiment of the present invention, the functionality ofheating element 212 and 226 and temperature sensors 214 and 228 can beaccomplished by the thin film heaters 112.

In one embodiment, the system 200 sends power to the thin-film heaters112 a and/or 112 b, thereby causing them to heat up, based on a controlsignal sent by the PCR zone temperature controller 210 or the thermalmelt zone temperature controller 224. The control signal can be, forexample, a pulse width modulation (PWM) control signal. An advantage ofusing a PWM signal to control the heaters 212 is that with a PWM controlsignal, the same voltage potential across the heaters may be used forall of the various temperatures required. In another embodiment, thecontrol signal could utilize amplitude modulation or alternatingcurrent. It may be advantageous to use a control signal that isamplitude modulated to control the heaters 212 because a continuousmodest change in voltage, rather than large voltage steps, avoids slewrate limits and improves settling time. Further discussion of amplitudemodulation can be found in U.S. Patent Application Publication No.2011/0048547, which is incorporated herein by reference in its entirety.In some embodiments, the desired temperature for the heaters is reachedby changing the duty cycle of the control signal. For example, in onenon-limiting embodiment, the duty cycle of the control signal forachieving 95° C. in a PCR heater might be about 50%, the duty cycle ofthe control signal for achieving 72° C. in a PCR heater might be about25%, and the duty cycle of the control signal for achieving 55° C. in aPCR heater might be about 10%. In other embodiments, other duty cyclesmay be used as would be apparent to persons skilled in the art.

The microfluidic device 100 and the system 200 can be used inconjunction with aspects of the present invention. For example, thesystem 200 may calibrate the thermal control elements 112 of themicrofluidic device 100 using an amplicon having a known or expectedthermal melting temperature, in accordance with aspects of theinvention. However, an amplicon having a known or expected thermalmelting temperature may be used to calibrate thermal control elements inmicrofluidic devices and systems other than the microfluidic device 100and the system 200 illustrated in the FIGS. 1 and 2.

In high resolution thermal melting instruments, such as system 200,temperature precision is of extreme importance. For example, a smallbias or shift in temperature may cause a genotype misclassification whenanalyzing melt curves. Whether analyzing fluorescence versus temperatureor derivative of fluorescence with respect to temperature, a homozygousDNA sample containing a single nucleotide polymorphism and a wildtypeDNA sample have the same shape and only differ by a small temperatureshift. For example, FIG. 18A illustrates fluorescence versus temperaturemelt curves for Warfarin CyP2C9*2, and FIG. 18B illustrates thederivative of fluorescence with respect to temperature melt curves forWarfarin CyP2C9*2. As shown in FIG. 18B, the temperature differencebetween the two profiles, or average difference in true meltingtemperature, is only 0.4° C. For such genotypes that exhibit the sameshape and only differ by a shift, the melting temperature of the DNA,T_(m), estimated by the peak temperature of the derivative is often usedto classify the genotypes. A small drift or bias in measured temperaturemay increase the misclassification of these genotypes. If there is apositive temperature shift or drift, a homozygous mutant can bemisclassified as a wildtype. Conversely, if there is a negativetemperature shift or drift, a wildtype can be misclassified ashomozygous mutant.

A temperature shift may be due to spatial differences in temperaturesuch as in a well plate (gradient), or a shift in time (drift). In anideal system, where there are no temperature shifts, or biases inmeasured temperature in space or time, there would be a much lowervariance in melting temperatures for a particular genotype. However, inreal instruments, temperature gradients and drift are real phenomena.

One aspect of the present invention is the calibration of thetemperature of a microfluidic channel (e.g., channel 102) of amicrofluidic device (e.g., device 100). In some embodiments, thecalibration may be performed by calibrating one or more thermal controlelements (e.g., thin film resistive heaters 112) that control and/ordetect the temperature of a microfluidic channel. In some embodiments,the temperature of a plurality of microfluidic channels (e.g., channel102) may be calibrated. In some embodiments, the calibration may beperformed using a single amplicon having a known or expected thermalmelting temperature as a calibrant. The amplicon is introduced into thethermal melting zone of the microfluidic channels and the amplicon ismelted. In one embodiment, the amplicon may be derived from genomic DNA.In one particular embodiment, the amplicon may be produced from an UltraConserved Element (UCE). In an alternative embodiment, the amplicon maybe produced from a synthetic DNA. In a further embodiment, the ampliconmay be produced from coding sequences, such as a sickle cell codingsequence.

In some embodiments, the calibration may be performed using a two ormore amplicons each having a known or expected thermal meltingtemperature. In particular embodiments, the two or more amplicons may beproduced from genomic DNA, synthetic DNA or a combination of one or moreamplicon derived from genomic DNA and one or more synthetic amplicons.In addition, one or more of the two or more amplicons may be UCEs.

FIG. 3A illustrates a process 300 a for calibrating the temperature of amicrofluidic channel of a microfluidic device in accordance with anembodiment of the present invention. The process 300 a may begin at step302 at which amplicons are introduced into the microfluidic channel. Ina preferred embodiment, each one of the one or more amplicons has aknown thermal melting temperature. In other words, in a preferredembodiment, the temperature at which each of the one or more ampliconsis expected to melt is known. In one embodiment, the one or moreamplicons comprise any synthetic sequence.

At step 304, a thermal melt analysis may be performed on theamplification product. In other words, the amplicons may be melted, andthe temperature at which the amplicons melt may be measured. In thisway, the process 300 a may determine the temperature at which each oneof the one or more amplicons in a microfluidic channel melted. In oneembodiment, the thermal melt analysis may be performed by exposing theamplicons to a temperature ramp. The amplicons may be exposed to thetemperature ramp (e.g., heating the thermal control element at, forexample, 0.5 degrees Celsius per second over a range of temperaturesthat includes temperatures corresponding to the features of the thermalresponse profiles of the amplicons) in a thermal melt zone of amicrofluidic device (e.g., thermal melt zone 106 of microfluidic device100). During the thermal ramp, a dependent variable (e.g., fluorescenceintensity) of the amplicons and the measurement value of the thermalcontrol element is monitored to generate a thermal response profile. Thetemperature may be measured by a thermal melt zone fluorescencemeasurement system (e.g., thermal melt zone fluorescence measurementsystem 232). However, any known method and/or device for performingthermal melt analysis may be used.

In one embodiment, thermal melt analysis may be an analysis of adependent variable related to a solution (e.g., reagent solution) thatis subject to a thermal variation, i.e., the relationship between asolution's temperature and the dependent variable. In some embodiments,the thermal response profile may be a melt curve, i.e. the fluorescentmelt analysis of a solution to determine the relationship between theamount of fluorescence and the solution's temperature. In someembodiments, generating such a thermal response profile can includeloading a microfluidic chip (e.g., the microfluidic chip 100) into asystem for controlling reactions in the microfluidic chip (e.g., system200), loading a droplet, plug, slug, or continuous flow of the reagentsincluding the one or more amplicons into the chip (e.g., into amicrofluidic channel 102), and controlling a thermal control element(e.g., heater 112) that is in thermal communication with the ampliconsto heat the amplicons while monitoring the temperature of the thermalcontrol element and while monitoring the dependent variable (e.g.,fluorescence in the case in which a melt curve is obtained) of theamplification product.

In one embodiment, the apparent (i.e., measured) temperatures of eachfeature (e.g., the apparent melt temperature corresponding to eachamplicon) may be identified from a thermal response profile. In someembodiments, the apparent temperatures may be determined from thederivative plot using peak-picking. In other embodiments, the apparenttemperatures may be determined by cross-correlating the derivative plotwith a known template (i.e., an expected thermal response profile) foreach amplicon, or shifting and stretching the thermal response profileto match a predefined template.

At step 306, for each of the one or more amplicons, the measuredtemperature at which the amplicon melted is compared to the known (i.e.,expected) thermal melt temperature for the amplicon. The comparison maybe performed by, for example, a digital or analog comparator. Thecomparator may be part of a control system of a microfluidic system(e.g., control system 250 of system 200). However, any known methodand/or device for performing the comparison may be used.

At step 308, the temperature of the microfluidic channel is calibratedbased on the results of the comparison(s) of the measured melttemperature(s) to the respective known thermal melt temperature(s). Thecalibration may be performed by adjusting a controller (e.g., controlsystem 250) and/or one or more thermal control elements (e.g., heater112) that control and/or detect the temperature of the microfluidicchannel (e.g., channel 102).

In some embodiments, calibration of a thermal control element caninclude determining the coefficients of a calibration equation, i.e., anequation that models the relationship between the temperature of thethermal control element and a measurement value. In an embodiment, thatmeasurement value may be an electrical characteristic of the thermalcontrol element. For example, a calibration equation for a resistiveheater 112 may model the relationship between the temperature of theheater 112 and the resistance of the heater 112. In this embodiment, thecalibration equation may model a linear relationship as shown inEquation 1:T=k ₀ +k ₁ R  Equation 1

In this case, T is temperature of the thermal control element, R is theresistance of the thermal control element, and k₀ and k₁ are constants(i.e., the coefficients) to be determined by calibration. Twocalibration coefficients can be determined by, for example, measuringthe resistance R at two known temperatures (T₁ and T₂). In some aspectsof the present invention, these measurements can be obtained by heatingthe thermal control element (e.g., a heater 112) in thermal contact witha droplet, plug, slug, or continuous flow of the reagents including oneor more ampliocns (e.g., while the reagents are in a microfluidicchannel 102 corresponding with the heater 112) and measuring theresistance of the thermal control element (e.g., measuring theresistance of the heater 112) at temperatures that coincide with two ormore features (e.g., melting points) of the thermal response profile ofthe amplicons.

In other embodiments, the calibration equation may model the relationbetween other electrical factors, such as the current, electricpotential, applied power, resistivity, conductivity, or other relatedquantities. In some aspects, the measurement value may be anindependently controlled aspect of the thermal control element that isrelated to the temperature of the thermal control element. In otheraspects, the measurement value could be any factor related to thetemperature of the thermal control element.

In some embodiments, the calibration equation may contain morecoefficients to be determined. For example, in the case where themeasurement value is resistance, some calibration equations can model aquadratic relationship as shown in Equation 2:T=k ₀ +k ₁ R+k ₂ R ²  Equation 2In this case, an amplification product resulting from reagents havingthree or more features in its thermal response profile may be preferableto more accurately determine all of the coefficients. Further, one ofordinary skill will comprehend that this approach may be expanded for acompound calibrator having n or more features, using Equation 3:T=k ₀ +k ₁ R+k ₂ R ² + . . . k _(n-1) R ^(n-1)  Equation 3

Furthermore, in some embodiments, more accurate values for thecoefficients may be obtained by utilizing reagents having more featuresthan there are coefficients to be determined (i.e., determine morecorrespondences between temperature and resistance of the thermalcontrol element than there are coefficients). The resultingover-determined system can be solved, for example, using the leastsquares method.

In some embodiments, the thermal control element can be calibrated usingan ambient temperature (i.e., room temperature) in addition to one ormore temperatures determined based on features (e.g., melting points) ofthermal response profiles. For example, calibration of the temperatureof the microfluidic channel may include determining the thermal controlelement's output (e.g., the resistance of the heater 112) while thethermal control element is at a known, ambient temperature. In someembodiments, the ambient temperature can be measured with a separatetemperature measurement device such as, for example, a precision RTD orthermocouple. However, any other suitable temperature measurement deviceincluding non-contact methods are appropriate as will be understood bythose skilled in the art.

In other embodiments, the one or more amplicons used to calibratetemperature in a microfluidic channel of a microfluidic device may besynthetic amplicons, i.e. amplicons produced from synthetic sequences.In a preferred embodiment, the amplicon(s) derived from syntheticsequences will not interfere with clinical samples and may be producedfrom a vector. In a preferred embodiment, the one or more calibrants aredesigned from a synthetic sequence will not interfere with clinicalsamples and may easily be produced from a vector. In some embodiments,the one or more sequences are chosen such that they are not present inhuman genomic DNA. In other embodiments, the one or more sequences arechosen such that they are not present in mammalian genomic DNA. Inadditional embodiments, the one or more sequences are chosen such thatthey are not present in vertebrate DNA. In one embodiment, one of theone or more amplicons is named RFCAL-100. In another embodiment, one ofthe one or more amplicons is named RFCAL-200. In certain embodiments,the one or more amplicons are cloned in a vector. Any suitable vectorwell known to the skilled artisan may be used, such as a plasmid, avirus, and the like, into which a synthetic sequence has been inserted.

In some embodiments of the present invention, one or more of the one ormore amplicons used to calibrate temperature in a microfluidic channelof a microfluidic device may be produced from UCEs. UCEs are involved intranscription regulation and in early development, and scientistsbelieve UCEs were functional introns common to all life forms in theevolutionary time. In the human genome, there are a large number ofUCEs, which may be sequences longer than 200 base pairs (bp). UCEs arealso conserved among vertebrates' genomes as well. UCEs are present inall human chromosomes, except for Y and 21 chromosomes. UCEs have a verylow mutation rate and have not changed at all during the last 300millions years or more.

Aspects of the present invention can be used to calibrate resistivesensors, thermistors, diode temperature sensors, thermocouples, or anyother suitable temperature measuring devices. The present invention canfurther be used to calibrate resistive sensors that are also used forheating, such as thin-film platinum elements (or nickel or copper or anyother material as would be understood by those skilled in the art).

Another aspect of the present invention is, for temperature shifts inspace and/or time that follow an observable pattern or trend, theapplication of a temperature correction to regenerate and/or replottemperature corrected melt curves and decrease the variance in meltingtemperatures. In other words, aspects of the present invention mayrecalibrate the temperature profile such that the measured temperaturesare closer to their true values. Accordingly, aspects of the presentinvention may be used to decrease the misclassification rate whengenotyping. In some embodiments, neighboring positive control DNAsamples (in space or time) of known melting temperatures may be used tocalculate and apply a temperature correction to DNA melt curves ofunknown genotypes. In some embodiments, positive control samples (i.e.,calibrants or amplicons having a known or expected melt temperature) maybe melted before and/or after and/or simultaneously with unknownsamples, and the derivative of their melt curves may exhibit one or twoor more peaks.

For example, FIG. 19A illustrates fluorescence versus temperature meltcurves for a calibrant (e.g., a 207 bp UCE sequence described in moredetail below) according to an embodiment of the invention on a wellplate based system, and FIG. 19B illustrates that the derivative offluorescence with respect to temperature melt curves for the UCEcalibrant exhibits a single peak. For another example, FIG. 20Aillustrates fluorescence versus temperature melt curves for a calibrant(e.g., a synthetic calibrant comprising RFCAL-100 and RFCAL-200described in more detail below) according to another embodiment of theinvention on a microfluidic channel based system, and FIG. 20Billustrates that the derivative of fluorescence with respect totemperature melt curves for the calibrant exhibits two peaks (i.e., onepeak around 70° C. and a second peak around 90° C.). Aspects of thepresent invention may use a calibrant producing a melt curve having aderivate that exhibits one or two peaks, such as those shown in FIGS.19B and 20B, to calibrate and apply a temperature correction to DNA meltcurves of unknown genotypes.

One aspect of the present invention is a method of calibrating thetemperature of a thermal melt in a microfluidic channel present in amicrofluidic system. The method may comprise: (a) introducing into themicrofluidic channel reagents comprising a test sample containing humangenomic DNA, at least a pair of primers for one or more nucleic acids ofinterest and a pair of primers for a calibrant, wherein the calibrant isfound in genomic DNA; (b) amplifying the genomic DNA to produce acalibrant amplicon and an amplicon of the at least one nucleic acid ofinterest; (c) melting the amplicons and determining a meltingtemperature of each of the amplicons; (d) comparing the determinedmelting temperature of the calibrant amplicon with an expected meltingtemperature of the calibrant amplicon; and (e) calibrating the meltingtemperature of the amplicon of the at least one nucleic acid of interestbased on the melting temperature of the calibrant amplicon.

FIG. 3B illustrates a process 300 b for calibrating the temperature of athermal melt in a microfluidic channel of a microfluidic device inaccordance with an embodiment of the present invention. The process 300may begin at step 312 at which reagents are introduced into themicrofluidic channel. The reagents may include a test sample, a pair ofprimers for a nucleic acid of interest and/or a pair of primers for acalibrant. In some embodiments, the reagents may also include one ormore additional pairs of primers for one or more additional nucleicacids of interest. In one embodiment, the test sample may contain humangenomic DNA. However, the test sample may contain other types of DNA inaddition to or as an alternative to human genomic DNA. In someembodiments, the calibrant is found in genomic DNA, such as, forexample, human genomic DNA. In one non-limiting embodiment, thecalibrant is a UCE. However, in other embodiments, the calibrant may bea synthetic calibrant. In some embodiments, such as embodiments wherethe calibrant is a UCE, the calibrant may be included in the testsample. In other embodiments, such as embodiments where the calibrant issynthetic, the reagents additionally include the calibrant. In someembodiments, the reagents may include one or more additional pairs ofprimers for one or more additional calibrants, which may also beincluded in the reagents. In a preferred embodiment, each one of the oneor more calibrants has a known thermal melting temperature. In otherwords, in a preferred embodiment, the temperature at which each of theone or more amplicons is expected to melt is known. Step 312 may includethe reagents being introduced in separate fluid segments, such that theprimers for the nucleic acids of interest may be in a separate fluidsegment than the primers for the calibrant. A fluid segment containingthe primers for the calibrant may be introduced before and aftermultiple fluid segments containing primers for the nucleic acid ofinterest, or the fluid segment containing the primers for the calibrantmay be alternately introduced with fluid segments containing the primersfor the nucleic acids of interest, such that a fluid segment containingthe primers for the calibrant may be present before and after each fluidsegment containing primers for the nucleic acids of interest.

At step 314, the reagents, which may include the test sample, at least apair of primers for one or more nucleic acids of interest and at leastof pair of primers for one or more calibrants are amplified.Amplification of the reagents in step 314 may produce an amplificationproduct comprising one or more calibrant amplicons and an amplicon ofthe one or more nucleic acids of interest. In one embodiment, theamplification may be performed by exposing the reagents to a PCRtemperature cycle a plurality of times to effect PCR amplification. Thereagents may be exposed to the PCR temperature cycle in a PCR zone of amicrofluidic device (e.g., PCR zone 104 of microfluidic device 100).However, any known method and/or device for amplication of reagents maybe used.

At step 316, a thermal melt analysis may be performed on theamplification product. In other words, the one or more calibrantamplicons and an amplicon of the one or more nucleic acids of interestmay be melted, and the temperature at which the amplicons melt may bemeasured. In this way, the process 300 b may determine the temperatureat which each of the amplicons in the amplification product in amicrofluidic channel melted. In one embodiment, the thermal meltanalysis may be performed by exposing the amplicons to a temperatureramp. The amplicons may be exposed to the temperature ramp (e.g.,heating the thermal control element at, for example, 0.5 degrees Celsiusper second over a range of temperatures that includes temperaturescorresponding to the features of the thermal response profiles of theamplicons) in a thermal melt zone of a microfluidic device (e.g.,thermal melt zone 106 of microfluidic device 100). During the thermalramp, a dependent variable (e.g., fluorescence intensity) of theamplicons and the measurement value of the thermal control element ismonitored to generate a thermal response profile. The temperature may bemeasured by a thermal melt zone fluorescence measurement system (e.g.,thermal melt zone fluorescence measurement system 232). However, anyknown method and/or device for performing thermal melt analysis may beused.

In one embodiment, thermal melt analysis may be an analysis of adependent variable related to a solution (e.g., reagent solution) thatis subject to a thermal variation, i.e., the relationship between asolution's temperature and the dependent variable. In some embodiments,the thermal response profile may be a melt curve, i.e. the fluorescentmelt analysis of a solution to determine the relationship between theamount of fluorescence and the solution's temperature. In someembodiments, generating such a thermal response profile can includeloading a microfluidic chip (e.g., the microfluidic chip 100) into asystem for controlling reactions in the microfluidic chip (e.g., system200), loading a droplet, plug, slug, or continuous flow of the reagentsincluding the one or more amplicons into the chip (e.g., into amicrofluidic channel 102), and controlling a thermal control element(e.g., heater 112) that is in thermal communication with the ampliconsto heat the amplicons while monitoring the temperature of the thermalcontrol element and while monitoring the dependent variable (e.g.,fluorescence in the case in which a melt curve is obtained) of theamplification product.

In one embodiment, the apparent (i.e., measured) temperatures of eachfeature (e.g., the apparent melt temperature corresponding to each ofthe one or more calibrant amplicons and/or each of the amplicon of theone or more nucleic acids of interest) may be identified from a thermalresponse profile. In some embodiments, the apparent temperatures may bedetermined from the derivative plot using peak-picking. In otherembodiments, the apparent temperatures may be determined bycross-correlating the derivative plot with a known template (i.e., anexpected thermal response profile) for each amplicon, or shifting andstretching the thermal response profile to match a predefined template.

At step 318, for each of the one or more calibrant amplicons, themeasured temperature at which the calibrant amplicon melted is comparedto the known (i.e., expected) thermal melt temperature for the calibrantamplicon. The comparison may be performed by, for example, a digital oranalog comparator. The comparator may be part of a control system of amicrofluidic system (e.g., control system 250 of system 200). However,any known method and/or device for performing the comparison may beused.

At step 320, the melting temperature(s) of the amplicon of the one ormore nucleic acids of interest of the microfluidic channel is calibratedbased on the measured melt temperature(s) of the one or more calibrantamplicons. For example, the melting temperature(s) of the amplicon ofthe one or more nucleic acids of interest of the microfluidic channel iscalibrated based on the results of the comparison(s) of the measuredmelt temperature(s) of the one or more calibrant amplicons to therespective known thermal melt temperature(s). In one non-limitingembodiment, the calibration is performed by shifting the melttemperature(s) of the amplicon of the one or more nucleic acids ofinterest in accordance with the results of the comparison(s) of themeasured melt temperature(s) of the one or more calibrant amplicons tothe respective known thermal melt temperature(s).

As noted above, in some embodiments, the microfluidic channel reagentsinclude a pair of primers for one nucleic acid of interest. As usedherein, nucleic acid of interest refers to a nucleic acid whose presenceor absence is to be determined in a test sample. In other embodiments,the microfluidic channel reagents include a pair of primers for each oftwo or more nucleic acids of interest. In some embodiments, thecalibrant found in genomic DNA is a DNA segment that is known to bepresent in all genomic DNA of interest, such as human genomic DNA. Inother embodiments, the calibrant is a UCE. In some embodiments, themicrofluidic channel reagents may contain a pair of primers for thecalibrant. In certain embodiments, the amplification of the microfluidicreagents produce a calibrant amplicon. In certain embodiments, theamplification of the calibrant does not affect amplification of the oneor more nucleic acids of interest.

In some embodiments, the determining the melting temperature of each ofthe amplicons may comprise: obtaining a derivative of a melt curve ofthe melting of the amplicons using a Savitzky-Golay filter, and findingthe temperatures at the maximum negative derivatives of the derivativeof the melt curve. The determining the melting temperature of each ofthe amplicons may comprise: cross-correlating peaks of a derivative of areference melt curve against peaks of a derivative of a melt curve ofthe melting of the amplicons, and selecting the temperatures having thehighest correlation as the melting temperatures of the amplicons. Thecomparing the determined melting temperature of the calibrant ampliconwith the respective expected melting temperature of the calibrantamplicon may comprise calculating a slope correction factor and anintercept correction factor using the respective expected meltingtemperature of the calibrant amplicon. Calibrating the meltingtemperature of each of the amplicons of the nucleic acids of interestmay comprise calculating an adjusted temperature using the calculatedslope correction factor and the calculated intercept correction factorof the calibrant amplicon.

The method may comprise using the comparison of the determined meltingtemperature of the calibrant amplicon with the respective expectedmelting temperature of the calibrant amplicon to validate amplificationof the one or more amplicons of the one or more nucleic acids ofinterest. The validation may comprise determining the corrected meltingtemperatures of the amplicons of the nucleic acids of interest.

In one embodiment, thermal melt analysis may be an analysis of adependent variable related to a solution (e.g., reagent solution) thatis subject to a thermal variation, i.e., the relationship between asolution's temperature and the dependent variable. In some embodiments,the thermal response profile may be a melt curve, i.e. the fluorescentmelt analysis of a solution to determine the relationship between theamount of fluorescence and the solution's temperature. In someembodiments, generating such a thermal response profile can includeloading a microfluidic chip (e.g., the microfluidic chip 100) into asystem for controlling reactions in the microfluidic chip (e.g., system200), loading a droplet, plug, slug, or continuous flow of the reagentsincluding test sample and primers into the chip (e.g., into amicrofluidic channel 102) amplifying the calibrant and nucleic acid ofinterest, and controlling a thermal control element (e.g., heater 112)that is in thermal communication with the amplification product to heatthe amplification product while monitoring the temperature of thethermal control element and while monitoring the dependent variable(e.g., fluorescence in the case in which a melt curve is obtained) ofthe amplification product.

In one embodiment, the apparent (i.e., measured) temperatures of eachfeature (e.g., the apparent melt temperature corresponding to eachamplicon) may be identified from a thermal response profile. In someembodiments, the apparent temperatures may be determined from thederivative plot using peak-picking. In other embodiments, the apparenttemperatures may be determined by cross-correlating the derivative plotwith a known template (i.e., an expected thermal response profile) foreach calibrant, or shifting and stretching the thermal response profileto match a predefined template.

In some embodiments of the present invention, one or more of the one ormore calibrants used to calibrate temperature of a thermal melt in amicrofluidic channel of a microfluidic device may be UCEs. Because UCEsare present in all genomic DNA, every time that an amplification is doneusing a genomic DNA, one or more specific Ultra Conserved Element (UCE)DNA sequences may also be amplified.

In some embodiments of the present invention, a pair of primers may bedesigned to amplify a specific UCE sequence that is used as a calibrant.Using an amplification process (e.g., step ***), such as PCR, the pairof primers may be used to make the same UCE amplicon over and over. TheUCE amplicon is will always shows the same melt temperature (T_(M)).Based on the characteristic of the UCE amplicons always showing the sameT_(M), the UCE smplicon can be used, for example, for calibratingmelting temperature in a microfluidic system having one or moremicrofluidic channels.

Accordingly, in one aspect, the present invention provides methods,devices, and systems for using a pair of primers for amplifying acalibrant found in human genomic DNA in PCR and thermal melt analysisdevices such that the calibrant amplicon is used to calibrate themelting temperature of an amplicon of a nucleic acid of interest. In oneembodiment, the UCE primers are used at the same time with at least apair of primers for one or more nucleic acids of interest, during PCRamplification and melting, the melt temperature (T_(M)) of an amplicongenerated from a nucleic acid of interest is correct. This may beaccomplished because the UCE primer product may have a known T_(M).

Embodiments of the present invention can be used in a variety ofinstruments but are particularly useful in PCR and thermal melt systemsthat perform in vitro diagnostics. Embodiments of the present inventionmay be used to calibrate sensors and heaters that are intended forthermal melt of samples (diagnostics) as well as other heaters andsensors within the instrument that perform entirely different functions(e.g., sample prep or PCR).

In another aspect, the present invention provides a multiple ampliconcalibrator system. In other words, reagents containing two or moreamplicons may be used to calibrate the temperature of a microfluidicchannel. For instance, a dual amplicon calibrator may be used togenerate accurate high resolution melting on certain microfluidic chipbased systems. In a non-limiting embodiment, one or more of the multipleamplicons in the multiple amplicon calibrator system are Ultra ConservedElements (UCEs). In another non-limiting embodiment, one or more of themultiple amplicons in the multiple amplicon calibrator system aresynthetic. In another non-limiting embodiment, one or more of themultiple amplicons are UCEs, and one or more of the multiple ampliconsare synthetic.

As described above, one embodiment of the present invention may utilizetwo amplicons. In one non-limiting example, one amplicon may be from adesired DNA sequence, and the other amplicon may be a UCE amplicon. Inone embodiment, the DNA sequence may be derived from the sequence of anyhuman gene or other sequence. One example of a gene that could be usedis a sickle cell gene. One example of a sequence for the sickle cellgene is a 444 bp fragment. In another embodiment, the UCE amplicon is a415 bp UCE amplicon. The melt temperature Tm for the sickle sequence isapproximately 84° C., and the melt temperature Tm for the UCE sequenceis 74° C. In this non-limiting example, both amplicons were derived byamplifying human genomic DNA. However, it is not necessary that bothamplicons in a dual amplicon calibrator system be derived by amplifyinghuman genomic DNA.

In certain embodiments, certain limitations may exist with the dualcalibrant system using the amplicon derived from the 444 bp fragment ofthe sickle gene and the 415 bp fragment of the UCE described above.First, the span covered by the two calibrators may not cover the fullrange of anticipated melt temperatures. Second, the amplicons werederived from genomic DNA and thus potentially could serve as a source ofcontamination for subsequent amplification reactions. Finally,manufacture of these calibrators may require amplification from humanDNA, which was obtained from DNA banks, such as the Coriell DNA banks.Thus, these calibrators may not represent material that would be easy tomanufacture or guarantee as a source.

Another aspect of the present invention is a dual amplicon calibratorsystem with a wide temperature span and two or more amplicons derivedfrom synthetic sequences. In a preferred embodiment, the amplicons aredesigned from synthetic sequences so that they may be readily produced.In some embodiments, the sequences are chosen such that they are notpresent in human genomic DNA. In other embodiments, the sequences arechosen such that they are not present in mammalian genomic DNA. Inadditional embodiments, such sequences are chosen such that they are notpresent in vertebrate DNA. In some embodiments, the sequences are chosento produce amplicons with a T_(M) span greater than 5 degrees Celsius,preferably greater than 10 degrees Celsius, and more preferably greaterthan 20 degrees Celsius. In one embodiment, a first amplicon is namedRFCAL-100. In another embodiment, a second amplicon is named RFCAL-200.In certain embodiments, the two or more amplicons are cloned in avector. Any suitable vector well known to the skilled artisan may beused, such as a virus, and the like.

Non-Limiting Calibrant Examples

Genomic UCE Sequence

In a non-limiting example of a calibrant that may be used to calibratethe temperature of a microchannel of a microfluidic device is a UCEsequence with 207 base pairs. In development, the 207 bp UCE sequencewas found using the Bioscience World web site www.bioscienceworld.ca.The sequence was then checked against the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) database. The 207 bp sequence (SEQ ID NO:1) was shown to be partof the human chromosome 17 sequence (GenBank accession numberAF287967.1). Using Primer 3 software, a set of two primers was designed(SEQ ID NO:2 and SEQ ID NO:3) which amplified a 71 bp product (SEQ IDNO:4).

Experiments using the UCE primers and different genomic DNA samplesshowed that all samples were amplified, and all samples amplified showthe same TMs. This experiment was done several times using the RocheLightCycler® 480 (LC480) cycler platform and a microfluidic instrument.The results of the experiments are shown in FIGS. 4 and 5. FIGS. 4 and 5illustrate a so-called “derivative plot” which describes the derivativeof fluorescence with respect to temperature as a function of temperature(e.g., dF/dT vs. T). FIG. 4 illustrates thermal melt curves of anamplification product generated using the UCE primers on themicrofluidic instrument and shows three different genomic DNA. FIG. 5illustrates thermal melt curves of an amplification product generatedusing the UCE primers on the LC480 cycler platform and shows sevendifferent genomic DNA. The results of the experiments demonstrate thatthere were not variations on the melt temperatures T_(M)s. Thus, theexperiments show that the UCE primers can be used to amplify UCEamplicons that act as temperature calibrators.

A second experiment was also performed. In the second experiment, a pairof primers for amplifying a nucleic acid of interest was loaded into thedevice and then subsequently the UCE primers were loaded into thedevice. The experiment was performed using a microfluidic instrument,and the results are shown in FIG. 6 and FIG. 7. FIG. 6 illustratesthermal melt curves showing three different genotypes of the primer ofstudy (i.e., a wildtype genotype (Wt), a homozygous genotype (Hm), and aheterozygous genotype (Ht)). FIG. 7 illustrates thermal melt curvesshowing the UCE amplicon of the UCE primers.

In the event that the amplicon of the UCE primers show variations inmelt temperatures T_(M)s, the amplification product of the primers ofstudy also may also show variation on the melt temperatures T_(M)s. Thismay indicate an impossible genotype differentiation between DNA samples.An example of results indicating an impossible genotype differentiationbetween DNA samples are shown in FIG. 8 and FIG. 9. FIG. 8 illustrates athermal melt curve showing three different genotypes, i.e. a wildtypegenotype (Wt), a homozygous genotype (Hm), and a heterozygous genotype(Ht). FIG. 9 illustrates a thermal melt curve showing the UCE ampliconof the UCE primers. As illustrated in FIG. 9, the melt temperaturesT_(M)s of the UCE amplicon of the UCE primers showed variation, and, asillustrated in FIG. 8, the melt temperatures of amplicon of the nucleicacid of interest showed the same variation.

To demonstrate that the UCE primers would not interfere with theamplification of a nucleic acid of interest and also help as atemperature calibrator, the following experiment was performed. The UCEprimers and primers for a nucleic acid of interest were added togetherwith the reaction mix components (i.e., reagents) and loaded into wellsof a 96-well plate. PCR amplification and melting analysis wereperformed using a LC480 cycler platform. The results are shown in FIGS.10 and 11. FIG. 10 illustrates a thermal melt curve showing the ampliconof the UCE primers added to the primers of study. FIG. 11 illustrates athermal melt curve showing the amplicon of the nucleic acid of interest.The results illustrated in FIGS. 10 and 11 confirm that the UCE primersdo not affect the amplification of the nucleic acid of interest. Inother words, the same melt temperatures were obtained for the ampliconof the nucleic acid of interest regardless of whether the UCE primerswere added with the primers of the nucleic acid of interest. The resultsalso show that the UCE amplicon can be used as temperature calibrator.

As shown by the experiments discussed above, in some embodiments, two ormore UCE primers may be used to provide a simple, fast and reliableprocedure to check temperature inside a microfluidic channel in amicrofluidic device or chip. Also, the experiments show that, in someembodiments, the two or more UCE primers may work as temperaturecalibrator when added following or ahead of the primers of study in achip. In this case, the UCE amplicons may be used to determine whetherthe microfluidic channels temperature did or did not reach the idealtemperature set, and, therefore, may be used to validate a PCR run. Theexperiments further show that the two or more UCE primers works in thesame fashion when used in a standard PCR instrument such as a LC480cycler platform. In some embodiments, the UCE primers may be added withtwo or more primers of study and may not interfere with theamplification product of the primers of study.

In other embodiments, the UCE primers may function as a positive controlto validate amplification has occurred. Using positive controls inbiological experiments is well known in the art. Positive controls aredefined as biological reactions where a specific phenomenon is expected.Specifically, the positive controls ensure that there is an effect whenthere should be an effect, by using an experimental treatment that isalready known to produce that effect. Positive controls are often usedto assess a success of a biological reaction. If a positive control doesnot produce an expected result, there may be something wrong with theexperimental procedure, the reagents or the equipment used, and theexperiment should be repeated. Accordingly, suitable control samplesinclude samples of a like or similar nature to a test sample but whichunlike the test sample have a known or expected characteristic.

As UCEs are present in all genomic DNA, every time that an amplificationis done using a genomic DNA, the UCE sequence is also amplified.Specifically, UCE primers are used to amplify UCE sequences to make thesame product repeatedly. Accordingly, amplification reactions performedon the UCE sequences can be used as a positive control in biologicalreactions. UCE presence in a biological test sample or in a separatecontrol sample will allow a researcher to determine whether a biologicalreaction has been successful based upon an outcome of the biologicalreaction performed on the control sample and/or a UCE related portion ofa reaction outcome for the test sample containing the UCE. Specifically,if the control sample containing UCE and UCE primers produces anexpected result, the biological reaction on the test sample isdetermined to have been successful. If the control sample produces anunexpected result, the biological reaction performed on the test sampleis determined to have been unsuccessful.

In one embodiment of the present invention, a testing device isconfigured to receive a biological sample and to run a biologicalreaction on the biological sample. A control sample and a test sample,each comprising reagents for the biological reaction, are provided to betested in the testing device. The test sample comprising a biologicalmaterial and the control sample comprising the biological material andreagents specific to one or more UCE are loaded into the testing deviceand the biological reaction is run separately on the test sample and onthe control sample. In yet another embodiment of the invention, the oneor more UCE reagents are added to the test sample and the biologicalreaction is run on the combined sample.

The test sample may comprise one or more test samples and test primerswhile the control sample may comprise test sample and UCE primers.Alternatively, the test sample is combined with the control sample andthe biological reaction is performed on the combined sample.Specifically, one or more UCE primers may work as a positive controlwhen added together with the test primers to the test sample. By way ofa non-limiting example, the biological reaction may be an amplificationreaction and/or a DNA analysis reaction. In this case, the UCE may beused to validate an amplification of the test amplicons. Theamplification of UCE indicates a successful sample preparation andsubsequent amplification. The one or more UCE primers added to the testsample in addition to the test primers do not interfere with theamplification product of the test primers. Accordingly, twoamplifications can be achieved simultaneously in one amplificationreaction. In this case, results of the amplification reactions for thetest amplicons and for the UCE may be separately monitored andevaluated. An expected amplification result of the UCE indicates asuccessful sample preparation and subsequent amplification. By way ofnon-limiting example, the amplification reaction may be a PCR reactionand/or polymerase-endonuclease amplification reaction. The nucleic acidanalysis reaction includes, but is not limited to a thermal meltanalysis, sequencing, separation methods including slab gel andcapillary electrophoresis (CE), fluorescence detection methods, shorttandem repeat analysis, and other analysis methodologies known to thoseof skill in the art. In one embodiment the control sample comprises allof the components of the test sample and UCE specific reagents.

In one embodiment of the invention, a controller is configured incommunication with the testing device to analyze a reaction result forthe test sample using the control sample as a positive control.Specifically, if amplification of UCE produces an expected result, theamplification of the test sample is determined to have been successful.To make a determination with respect to the UCE amplification result, adetection method may be used. In one embodiment, a thermal responseprofile, such as a melt curve, may be obtained. In such an instance, theUCE would have an expected melting temperature, and a melt curve showingthe expected melting temperature would be indicative of a positiveresult. An expected thermal response profile can serve as a positivecontrol to demonstrate that no contamination of the reagents or otheranomalies have occurred that would prevent amplification of the genomicnucleic acid. The control sample and the test sample can undergo thebiological reaction in series or in parallel. The device used to run thebiological reaction can be a microfluidic device or a non-fluidic devicesuch as a well plate loaded into a PCR machine.

In the illustrated embodiment shown in FIG. 1, the microfluidic device100 includes several microfluidic channels 102. The test sample and thecontrol sample may be loaded into the same channel or into differentchannels 102 of the microfluidic device 100. Each channel may besubdivided into a first portion extending through a PCR thermal zone 104and a second portion extending through a thermal melt zone 106. In oneembodiment, the test sample and the control sample may undergo anamplification reaction separately. In yet another embodiment, thebiological sample and UCE primers that would make up a separate controlsample may instead be added to the test sample to undergo anamplification reaction and a melt analysis in a single reaction. If theUCE amplification reaction exhibits expected results, the amplificationreaction performed on the test sample is determined to be successful,regardless of the actual presence of amplicons in that test sample. Inthis case, the reaction result may be reported and/or used for furtherprocessing. In contrast, if the UCE amplification shows unexpectedresults, the reaction performed on the test sample is determined to beunsuccessful. In this case, the experimental procedure associated withthe PCR and melting reaction may be readjusted and repeated. In yetanother embodiment of the present invention, there is provided apositive control formulation comprising one or more primers specific toa UCE. In one non-limiting embodiment, the positive control formulationcomprises one or more primers which have one or more of the sequencesshown in SEQ ID NOs 2 and 3.

Synthetic Calibrator

In one non-limiting embodiment of one or more amplicons that may be usedto calibrate the temperature of a microchannel of a microfluidic device,one or more of the one more amplicons may be synthetic amplicons. In anexemplary embodiment, the invention provides a dual amplicon calibratorsystem. The dual amplicon calibrator system may be used to improvecalibrator performance and production. In one non-limiting example, thedual amplicon calibrator system may be used in a high resolution DNAmelting instrument to improve calibrator performance and production.However, this is not required, and the dual amplicon calibrator systemmay be used in other systems. In some embodiments, the dual ampliconcalibrator system may have individual calibrators designed for the dualsystem. In one embodiment, testing of the dual amplicon calibratorsystem was performed on a LC480 cycling platform, and non-limitingexamples of possible formulations of the dual calibrator system weredeveloped.

In one non-limiting example, two amplicons that may be used calibratethe temperature of a microfluidic channel in a microfluidic device arereferred to herein as “sequence A” and “sequence B.” Sequence A is 100base pairs in length (SEQ ID NO:5), and sequence B is 200 base pairs inlength (SEQ ID NO:6). Although a particular example is described,different sequences and sequences having different lengths may also beused in embodiments of the present invention.

In development of the non-limiting example of a dual calibrator inaccordance with embodiments of the present, synthetic DNA constructs forthe two calibration points were used to allow specific targeting ofmelting temperatures TMs. Two DNA (i.e., sequence A and sequence B)sequences were created to work as a DNA template. The sequences weregenerated using a web-based program called ‘Random DNA SequenceGenerator”.

The two sequences were checked for similarity using BLAST program fromNCBI, After no similarities were found with any sequence on thedatabase, primers were targeted to the last 20 or 22 base pairs of thecalibrator sequences. Primers for Sequence A are those set forth in SEQID NO:7 and SEQ ID NO:8. Primers or Sequence B are those set forth inSEQ ID NO:9 and SEQ ID NO:10. To check for primer specificity, theUniversity of California, Santa Cruz, (UCSC) Genome Browser was used.The primers were verified to amplify only sequence A and sequence B.

After this preliminary work was done, a sequence was constructed andordered for both sequences using Gene Oracle clone services. Eachconstruct was separately cloned into a pGOv4 vector. The pGOv4 vector isa pUC based vector which is Ampicillin and Kanamycin resistant. ThepGOv4 map is shown in FIG. 12.

After the sequences were incorporated into a vector, the vectors wereRFCAL-100 which contains Sequence A and RFCAL-200 which containsSequence B. The sequence for RFCAL-100 is set forth in SEQ ID NO:11. Thesequence for RFCAL-200 is set forth in SEQ ID NO:12.

All primers were ordered high-performance liquid chromatography (HPLC)purified at 200 nM from Invitrogen. However, primers of differentpurifications and/or from different providers may be used. The primerswere then resuspended in PCR clean water at 100 uM. Genomic DNA was usedas a control DNA. For example, VKORC1 wild type from Paragon Dx may beused as a control DNA. Tables below provide additional details regardingthe reagents mixes and reagents catalog numbers.

TABLE 01 PCR reagents Reagent Manufacture Catalog # Taq polymeraseClontech Takara RR006L dNTPs Fisher Scientific AB-1124 PCR water Mo Bio17000-10 MgCl2 Ambion AM9530G LC Green Plus Idaho TechnologyBCHM-ASY-000 2× CULS Buffer CULS CULS0017 Genomic DNA control Paragon Dx004-GGCCGG

The reagents described above were used in the following concentrationsdepicted on tables below to create a DNA Reagent Mix, a Taq PolymeraseReagent Mix and a Primer Reagent Mix.

TABLE 02 DNA mix DNA Reagent Mix Volume 1 Rx Channel conc. Stock conc.DNA  1.8 uL 1 cp/nL 50 ng/uL 2× CULS Buffer  7.5 uL 1× 2× Alexa   3 uL10 uM 50 uM Water 2.69 uL — — Total   15 uL — —

TABLE 03 CR mix Taq Polymerase Reagent Mix Volume 1 Rx Channel conc.Stock conc. Enzyme 3.3 uL 0.25 U/uL 5 U/uL dNTPs   1 uL 0.37 mM 25 mM 2×CULS Buffer   5 uL 1× 2× Water 0.7 uL — — Total  10 uL — —

TABLE 04 Primer mix Primer Reagent Mix Volume 1 Rx Channel conc. Stockconc. Forward primer 0.4 uL 1 uM 100 uM Reverse primer 0.4 uL 1 uM 100uM 2× CULS Buffer   5 uL 1×  2× MgCl2 0.1 uL 3 mM 1000 mM LC Green Plus  4 uL 1× 10× Water 0.1 uL — — Total  10 uL — —

In this example, a total of 20 uL of a sum of all three reagent mixeswere added in a well of a 96-well plate. Each primer was added induplicate. The volume and percentage of each reagent mix is shown in thetable below.

TABLE 05 Volume and proportion of reagents per well Reagents per wellReagent Mix Volume Percentage DNA 12 uL  60% Polymerase  3 uL  15%Primer  5 uL  25% Total 20 uL 100%

All three groups of reagents were ordered from the laboratory's in-housestock supply. DNA and primers were added subsequently on theirrespective mixes.

In this example, primers were added in duplicates. Positive control wasadded (e.g., VKORC1 primers plus control DNA (VKORC1)) as well as aNegative control (e.g., VKORC1 primers plus 2×CULS buffer).

Reactions were amplified on the Roche LC480 cycling platform. The PCRprotocol used is shown on table below.

TABLE 07 PCR protocol PCR Protocol Pre Amplification 95° C./1 min  1×Amplification 95° C./5 sec 40× 38° C./5 sec 72° C./5 sec PosAmplification 37° C./10 sec  1× 95° C./10 sec Melt 55° C.-95° C./15 sec 1×

Annealing temperature at 38° C. worked well for both primers set.

FIG. 13 illustrates a graph depicting the calibrators' productamplification on a LC480 cycler platform. As illustrated in FIG. 13, thesamples were successfully amplified. RFCAL-200 DNA showed very strongfluorescence values. RFCAL-100 showed lower fluorescence values comparedwith RFCAL-200 as expected due to the sequence size and GC content.VKORC1 was used as a PCR positive control.

Melting peaks for both DNA samples also were successfully obtained. Bothpresented a single and defined peak that was far apart from each other.RFCAL-100 has a melt temperature T_(M) around 70° C., and RFCAL-200 hasa melt temperature T_(M) around 90° C. FIG. 14 illustrates thermal meltcurves depicting the amplification product of the calibrators melting ona LC480 cycler platform.

An aliquot of the PCR product was then checked for target sizeconfirmation using an Agilent Bioanalyzer 2100 microfluidics-basedanalysis platform. FIG. 15 illustrates the RFCAL-100 analysis results,and FIG. 16 illustrates the RFCAL-200 results.

Expected and observed band size for RFCAL 100 and RFCAL-200 PCR productis shown below.

Seq. product Bp expected Bp observed RFCAL-100 100 bp 113 bp RFCAL-200200 bp 204 bp

In a preferred embodiment, RFCAL-100 and RFCAL-200 are added on a 2:1ratio because RFCAL-100 presents a lower fluorescent values thanRFCAL-200. By doing so, RFCAL-100 and RFCAL-200 achieve roughfluorescence equivalence in the final formulation. FIG. 17 illustratesan RFCAL-melt profile for RFCAL-100 and RFCAL-200 on Idaho TechnologiesHR-1 high-resolution melter with the calibrator formulated in a 2:1ratio of RFCAL100 to RFCAL-200. As shown in FIG. 17, the fluorescentvalues of RFCAL-100 and RFCAL-200 are substantially the same at theseratios. However, it is not necessary that RFCAL-100 and RFCAL-200achieve rough fluorescence equivalence, and other ratios may be used.

In the exemplary embodiment, the two constructs and respective primersworked as desired. The primers were able to demonstrate a successfulamplification of the expected product. Each of their amplificationproducts showed a single peak with high fluorescent values upon melt.Product specificity was confirmed by demonstration of expected fragmentsizes on the Bioanalyzer microfluidics-based analysis platform.RFCAL-100 and RFCAL-200 are thus useful for temperature calibrationpurposes given their performance.

Although the specific examples of UCE and synthetic calibrants have beendescribed above, other calibrants may be used in accordance with aspectsof the present invention.

Embodiments of the present invention have been fully described abovewith reference to the drawing figures. Although the invention has beendescribed based upon these preferred embodiments, it would be apparentto those of skill in the art that certain modifications, variations, andalternative constructions could be made to the described embodimentswithin the spirit and scope of the invention.

Non-Limiting Temperature Calibration/Correction Examples

As noted above, the computation from a resistance measurement, R, of aheating sensing element to measured temperature, T_(mes), may take theform of the linear equation:T _(mes) =k ₁ R+k ₀  Equation 4

An error in terms k₁, or k₀ would lead to a bias or shift in themeasured temperature relative to the true temperature. Calibrants (i.e.,positive control DNA having a known or expected melt temperature) may beused to calculate the temperature compensation to shift the measuredtemperature closer to the true value.

Single Peak Temperature Calibration with Interpolation

When a single peak amplicon (e.g., derived from the 207 bp UCE sequence)is used as a calibrant (i.e., positive control), a single temperatureshift (correction in intercept k₀) may be applied to the span of allmeasured temperatures of a particular melt. In one embodiment, thisshift correction value k₀′ may be applied to an unknown DNA sample asfollows. First, the peak temperatures of a pre UCE melt (T_(m)^(pre UCE)) and a post UCE melt (T_(m) ^(post UCE)) are obtained.Second, the adjusted temperature of an assay melt that is run in betweenthe two UCEs (i.e., calibrants) is calculated by linear interpolation.

The peak temperatures of the pre UCE amplicon melt (T_(m) ^(pre UCE))and the post UCE amplicon melt (T_(m) ^(post UCE)) may be obtained, forexample, in step 304 of the process 300 a for calibrating thetemperature of a microfluidic channel of a microfluidic device shown inFIG. 3A or in step 316 of the process 300 b for calibrating thetemperature of a thermal melt shown in FIG. 3B. In other embodiments,the peak temperatures of the pre UCE amplicon melt (T_(m) ^(pre UCE))and the post UCE amplicon melt (T_(m) ^(post UCE)) may be obtained usingany suitable approach. For example, one suitable approach is a peakpicking approach that obtains T_(m) ^(pre UCE) and T_(m) ^(post UCE) by(i) using a Savitzky-Golay filter with a predefined window size,polynomial and temperature resolution (e.g., 2° C., 2^(nd) order, and0.01° C. respectively) to obtain the negative derivatives of the pre andpost UCE amplicon melts and (ii) finding the temperatures at the maximumnegative derivatives of the pre and post UCE amplicon melts. Here, thetemperatures at the maximum negative derivatives of the pre and post UCEamplicon melts are T_(m) ^(pre UCE) and T_(m) ^(post UCE).

In another embodiment, the peak temperatures of the pre UCE ampliconmelt (T_(m) ^(pre UCE)) and the post UCE amplicon melt (T_(m)^(post UCE)) may be obtained using a maximal correlation approach. Underthe maximal correlation approach, T_(m) ^(pre UCE) and T_(m) ^(post UCE)are obtained by using the derivative of the melt curve of a calibrant(i.e., positive control) (e.g., shown as a solid line in FIG. 21A) froma calibrated instrument as a reference to which a derivative of a meltcurve of the calibrant from one or more subsequent experiments (e.g.,shown as a dotted line in FIG. 21A) are shifted. Accordingly, y(x) forthe experimental calibrant may be shifted by various dx:y′(x)=y(x−dx)  Equation 5

In this way, the negative derivative of the reference calibrant (e.g.,shown as a solid line in FIG. 21A) may be correlated against the shiftedcalibrant of a subsequent experiment, y′(x), as shown in FIG. 21A. Theoptimal shift may be given by the dx that maximizes the correlationcoefficient as shown in FIG. 21B. In FIG. 21A, the optimal shift resultsin the dashed line. T_(m) ^(pre UCE) and T_(m) ^(post UCE) may then becalculated by subtracting the optimal dx from the T_(m) of the referenceUCE. This maximal correlation approach to calculating shifts in T_(m)may be more robust to noise than the picking the peak alternative, buteither method may be used.

Once T_(m) ^(pre UCE) and T_(m) ^(post UCE) are obtained, the adjustedtemperature of an assay melt that is run in between the two UCEs may becalculated by linear interpolation as follows:

$\begin{matrix}{{{\Delta\; T_{m}^{{Assay}\mspace{14mu}{minus}\mspace{14mu}{interpolated}\mspace{14mu}{UCE}}} = {T^{Assay} - T_{m}^{preUCE} + \frac{\begin{matrix}\left( {{melt\_ index}^{Assay} - {melt\_ index}^{preUCE}} \right) \\\left( {T_{m}^{postUCE} - T_{m}^{preUCE}} \right)\end{matrix}}{\left( {{{melt\_ index}\;}^{postUCE} - {melt\_ index}^{\mspace{11mu}{preUCE}}} \right)}}}{T^{{Assay}\;,{adjusted}} = {T_{m}^{{reference}\mspace{14mu}{UCE}} + {\Delta\; T_{m}^{{Assay}\mspace{14mu}{minus}\mspace{14mu}{interpolated}\mspace{14mu}{UCE}}}}}} & {{Equation}\mspace{14mu} 7}\end{matrix}$

Thus, the calculation for the shift correction value k₀′ is:

$\begin{matrix}{k_{0}^{\prime} = {T_{m}^{{reference}\mspace{14mu}{UCE}} - T_{m}^{{pre}\mspace{14mu}{UCE}} + \frac{\begin{matrix}\left( {{melt\_ index}^{Assay} - {melt\_ index}^{{pre}\mspace{14mu}{UCE}}} \right) \\\left( {T_{m}^{{post}\mspace{14mu}{UCE}} - T_{m}^{{pre}\mspace{14mu}{UCE}}} \right)\end{matrix}}{\left( {{melt\_ index}^{\;{{post}\mspace{14mu}{UCE}}} - {melt\_ index}^{\;{{pre}\mspace{14mu}{UCE}}}} \right)}}} & {{Equation}\mspace{14mu} 8} \\{\mspace{79mu}{and}\mspace{14mu}} & \; \\{\mspace{79mu}{T^{{Assay},{adjusted}} = {T^{assay} + k_{0}^{\prime}}}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

Note that in the equations above, T_(m) ^(reference UCE) is the true,known T_(m) of the UCE taken from the literature or measured from acalibrated instrument, and T_(Assay) is the assay temperature vector orT_(m) of the assay measured by the experimental instrument. Thecalculation of the shift correction value using Equation 8 may occur,for example, in step 306 of the process 300 a for calibrating thetemperature of a microfluidic channel of a microfluidic device shown inFIG. 3A or in step 318 of the process 300 b for calibrating thetemperature of a thermal melt shown in FIG. 3B. The calculation of theadjusted temperature shown in Equation 9 may occur, for example, in step308 of the process 300 a for calibrating the temperature of amicrofluidic channel of a microfluidic device shown in FIG. 3A or instep 320 of the process 300 b for calibrating the temperature of athermal melt shown in FIG. 3B.

FIG. 22A is a plot illustrating melting temperatures (Tms) of assays ofthe same genotype (open circles) and interlaced UCEs (solid pointsconnected by lines) collected from 7 different channels. FIG. 22B is aplot illustrating the standard deviation of the assays melted at thesame time across the 7 channels. FIG. 22C is a plot illustrating thechanges in the melt temperatures (ΔTms) of the assays relative to theinterpolated UCE melt temperature (Tm) using the immediately precedingand immediately following UCE. FIG. 22D is a plot illustrating thecorresponding standard deviation of the changes in the melt temperatures(ΔTms) shown in FIG. 22C. FIG. 22E is a plot illustrating the changes inthe melt temperatures (ΔTms) of the assays relative to the interpolatedUCE Tm using only the first and last UCE. FIG. 22F is a plotillustrating the corresponding standard deviation of changes in the melttemperatures (ΔTms) of the assays shown in FIG. 22E.

As shown in FIGS. 22A-22F, when there is a drift in temperature in aparticular channel (as shown by the drifting T_(m) of the UCEs) or ifthere is a shift in measured temperature from channel to channel,calculating the adjusted measured temperature of the assay relative tothe interpolated Tm of the pre and post UCEs may be used to help reducethe standard deviations of T_(m)s of the assays (of the same genotype)across the channels. In this way, calculating the adjusted measuredtemperature of the assay relative to the interpolated Tm of the pre andpost UCEs may be used to improve the accuracy of a genotype call.

In the example method using a single calibrant/peak set forth above, useof a genomic UCE (e.g., the 207 bp UCE sequence described above) is notnecessary. A synthetic sequence (e.g., RFCAL-100 or RFCAL-200) mayalternatively be used as the calibrant.

Dual Peak Temperature Calibration

When a dual peak calibrant (e.g., a calibrant comprising RFCAL-100 andRFCAL-200) is used as a positive control, a temperature scaling as wellas shift (correction in slope k₁ as well as intercept k₀) may be appliedto the span of all measured temperatures of a particular melt. For thisreason, using a dual peak positive control may lead to more accuratecompensation of measured temperature values. In one embodiment, thisscale correction k₁′, and shift correction value k₀′ may be applied toan unknown DNA sample as follows. First, the low and high peaktemperatures of an experimental dual peak calibrant (e.g., UCEand/orgenomic and/or synthetic) melt T_(m low) ^(UCE) and T_(m low)^(UCE) are obtained. Second, the adjusted temperature of an assay meltthat is run following the experimental positive control is obtained.

The low and high peak temperatures of an experimental dual peakcalibrant (e.g., UCE) melt may be obtained, for example, in step 304 ofthe process 300 a for calibrating the temperature of a microfluidicchannel of a microfluidic device shown in FIG. 3A or in step 316 of theprocess 300 b for calibrating the temperature of a thermal melt shown inFIG. 3B. The low and high peak temperatures of an experimental dual peakcalibrant (e.g., UCE) melt T_(m low) ^(UCE) and T_(m low) ^(UCE) may beobtained using any suitable approach. For example, one suitable approach(i) obtains the derivative of the melt curve of the experimentalcalibrant by using a Savitzky-Golay filter with a predefined windowsize, polynomial and temperature resolution (e.g., 2° C., 2^(nd) order,and 0.01° C. respectively) and (ii) finds the temperatures at themaximum negative derivatives (low and high) of the dual peak calibrant.Here, the temperatures at the maximum negative derivatives (low andhigh) of the dual peak calibrant are T_(m) ^(pre UCE) and T_(m)^(post UCE).

Another suitable approach for obtaining the low and high peaktemperatures of an experimental dual peak calibrant melt T_(m low)^(UCE) and T_(m high) ^(UCE) may use the derivative of a melt curve fora dual peak calibrant (i.e., positive control) obtained from acalibrated instrument as a reference. The low temperature and hightemperature peaks may be found (T_(m low) ^(UCE reference) andT_(m high) ^(UCE reference)) from the reference derivative of the meltcurve, and a window (e.g., ±1.5° C.) may be placed around each peak(shown by the dashed line segments of FIG. 23A).

The negative derivative of the left peak (dashed line portion of FIG.23A centered around 70° C.) of the reference derivative may becross-correlated against the negative derivative of the left peak of theexperimental calibrant shown in FIG. 23B. The two highestcross-correlation points may be obtained, and the temperature of thefirst (left) highest cross-correlation point may be taken as T_(m low)^(UCE). The negative derivative of the right peak (dashed line portionof FIG. 23A centered around 90° C.) of the reference derivative may becross-correlated against the negative derivative of the right peak ofthe experimental calibrant shown in FIG. 23B. The two highestcross-correlation points may be obtained, and the temperature of thesecond (right) highest cross-correlation point may be taken asT_(m high) ^(UCE).

Once T_(m low) ^(UCE) and T_(m high) ^(UCE) have been obtained, theadjusted temperature of an assay melt that is run before or followingthe experimental positive control may be obtained by calculating a slopecorrection factor and an intercept correction factor as follows:

$\begin{matrix}{{{slope\_ correction}{\_ factor}\text{:}\mspace{14mu} k_{1}^{\prime}} = \frac{\left( {T_{mhigh}^{UCEreference} - T_{mlow}^{UCEreference}} \right)}{\left( {T_{mhigh}^{UCE} - T_{mlow}^{UCE}} \right)}} & {{Equation}\mspace{14mu} 10} \\{{{intercept\_ correction}{\_ factor}\text{:}\mspace{14mu} k_{0}^{\prime}} = {T_{mhigh}^{UCEreference} - {k_{1}^{\prime}\left( T_{mhigh}^{UCE} \right)}}} & {{Equation}\mspace{14mu} 11}\end{matrix}$

The slope correction factor and the intercept correction factor may becalculated, for example, in step 306 of the process 300 a forcalibrating the temperature of a microfluidic channel of a microfluidicdevice shown in FIG. 3A or in step 318 of the process 300 b forcalibrating the temperature of a thermal melt shown in FIG. 3B.

Then, the adjusted temperature may be calculated from the slopecorrection factor and the intercept correction factor as follows:T ^(Assay,adjusted) =k ₁′(T ^(Assay))+k ₀′  Equation 12

Alternatively, these correction factors may be applied to the resistancevalues in real time to obtain the recalibrated temperature reading asfollows:T ^(Assay,adjusted) =k ₁ ′k ₁ R+k ₁ ′k ₀ +k ₀′  Equation 13

The calculation of the adjusted temperature shown in either Equation 12or Equation 13 may occur, for example, in step 308 of the process 300 afor calibrating the temperature of a microfluidic channel of amicrofluidic device shown in FIG. 3A or in step 320 of the process 300 bfor calibrating the temperature of a thermal melt shown in FIG. 3B.

As shown in FIG. 23C, using an out of calibration dual peak calibrant(e.g., a calibrant producing the dual peak melt curve shown in FIG.23B), the recalibration procedure described above may line up the lowand high peaks with the low and high peaks from the reference melt ofthe calibrator (e.g., the reference melt shown in FIG. 23A). Asillustrated in FIG. 23D, the subsequent melt of an independentcalibration check melt shows that this recalibration procedure works onsubsequent melts. FIG. 23E is a schematic diagram illustrating thefunctioning of the automatic calibration procedure using a dual peakcalibrant.

As shown in the temperature calibration examples set forth above,calibrants (i.e., positive control DNA) having known or expected melttemperatures and producing single or dual peak melt curves may be usedto intermittently account for calibration drift in the instrument.

What is claimed is:
 1. A method for providing a positive control in anucleic acid amplification reaction followed by a nucleic acid meltingusing ultra-conserved elements (UCE), the method comprising: providing acontrol sample, wherein the control sample comprises human genomic DNA,the human genomic DNA including a UCE sequence; providing a deviceconfigured to receive the control sample, wherein the device comprises asystem controller configured to control fluid flow, control temperaturefor the PCR reaction and control temperature for a thermal melt; addingone or more UCE specific primers to the control sample, wherein the oneor more UCE specific primers have the sequence shown in SEQ ID NOs 2 and3; loading the control sample into the device performing PCR to amplifythe UCE sequence; performing PCR on the control sample; following thePCR, performing a thermal melt analysis on the control sample to obtainmelting curves, wherein the system controller analyzes the melting curveand determines a melting temperature for the control sample; wherein thesystem controller analyzes a reaction result using the control sample asa positive control, wherein (i) if the control sample produces anexpected UCE melting temperature, the amplification reaction followed bythe thermal melt analysis is determined to have been successful; and(ii) if the control sample produces an unexpected UCE meltingtemperature, the amplification reaction followed by the thermal meltanalysis is determined to have been unsuccessful.
 2. The method of claim1, wherein the device is a microfluidic device comprising multiplemicrochannels and both performing the PCR and the thermal melt analysisfor the control sample occurs in a single channel.
 3. The method ofclaim 1, further comprising adjusting operating conditions and repeatingthe PCR followed by the thermal melt analysis if the reaction isdetermined to be unsuccessful.